Apoptosis is a naturally occurring cell death characterized by reduction in cell volume, nuclear condensation, cell blebbing, and endonucleolytic degradation of DNA at nucleosomal intervals. The onset of apoptosis arises from an activation of one or more genetic programs. Apoptosis is distinct from cell necrosis where chromatin clumps, organelles swell and eventually the cell membrane dissolves (Edgington, 1993).
The 19 kD (19K) protein coded by the adenovirus (Ad) E1B region confers a survival function in adenovirus-infected cells and prevents premature cell death. The DNA fragmentation observed in cells infected with E1B 19K mutants is reminiscent of that observed during apoptosis (Wyllie, 1980). Hence, one can infer that the 19 kD protein protects against a cell death program induced by viral infection, thus facilitating efficient virus replication. The E1A proteins, and specifically the conserved region (CR) 1 and CR2 which interact with cellular proteins p300 and pRb and induce cellular proliferation, have been implicated in the onset of the virus-induced death program (White et al., 1991; Mymryk et al., 1994).
The 19 kD protein suppresses the cytotoxic effects of certain external stimuli such as tumor necrosis factor (TNF)-.alpha. (Gooding et al., 1991; White et al., 1992) and anti-Fas antibody (Hashimoto et al., 1991). Both TNF-.alpha. and anti-Fas have been shown to cause cell death through apoptosis (Laster et al., 1988; Itoh et al., 1991; Watanabe-Fukunaga et al., 1992). Similarly, the 19 kD protein protects cells against the effects of DNA damaging agents such as the anti-cancer drug cisplatin (Subramanian et al., 1993) and UV (Tarodi et al., 1993). Both cisplatin (Sorenson & Eastman, 1988) and UV (reviewed by Williams, 1991) induce cell death through the apoptotic pathway.
Levels of p53 increase in response to DNA damaging agents and it appears that the accumulation of p53 is responsible for the induction of apoptosis by DNA damaging agents (Clarke et al., 1993; Lowe et al., 1993; Lane, 1993). Since 19K can efficiently suppress cell death induced by DNA damaging agents (Subramanian et al., 1993; Tarodi et al., 1993), it appears that 19K suppresses p53-dependent apoptosis (Debbas and White, 1993). Thus, the 19 kD protein provides a survival function in virus-infected cells and also protects cells against certain other cell death-inducing stimuli.
The survival function provided by E1B 19K appears to be strikingly similar to the activity of the cellular proto-oncogene, bcl-2. The bcl-2 oncogene enhances the survival of hematopoietic B and T cells by blocking apoptosis (Vaux et al., 1988; Sentman et al., 1991; Strasser et al., 1991). Overexpression of Bcl-2 protein inhibits apoptosis induced by treatment with glucocorticoids (Alnemri et al., 1992a), deprivation of cytokines (Nunez et al., 1990; Hockenbery et al., 1990), activation of the c-myc oncogene (Bissonnette et al., 1992; Fanidi et al., 1992), radiation (Strasser et al., 1991) and other DNA damaging agents (Tarodi et al., 1993).
Thus, Bcl-2 appears to protect against apoptosis induced by diverse agents. The effect of the 19 kD protein on cell death induced by such a multitude of stimuli has yet to be examined. However, it is known that the Bcl-2 protein can substitute for the 19 kD protein during adenovirus infection. The characteristic fragmentation of cellular DNA induced by infection with Ad2 19K mutants is suppressed in cells expressing the human Bcl-2 protein (Tarodi et al., 1993). Similarly, expression of Bcl-2 by an adenovirus 2/Bcl-2 recombinant virus does not induce DNA fragmentation in infected cells and forms small plaques on cell monolayers. Rao et al. (1992) reported that Bcl-2 can substitute for 19K, albeit at reduced levels, in transformation of primary rat kidney cells in cooperation with E1A.
Studies have indicated that there are a number of molecules with homology to and that have similar activities with bcl-2. Hence, there might be termed an assemblage of functionally related molecules which includes BHRF-1, bcl-X.sub.L, bcl-X.sub.s, mcl-1 and the like. The similarity can extend to a structural homology at the nucleic acid level, protein level or both. But as will be revealed hereinbelow, the homology need not be over the length of the molecule but can be confined to discrete functional portions of the molecule. Hence, under that criterion, 19K also comprises the assemblage of molecules relating to apoptosis.
The mechanism by which the 19K gene and the Bcl-2 protooncogene protect against cell death is not known. It is possible that those apoptosis-regulating proteins (hereinafter blocking proteins) mediate cell survival by interacting with certain cellular apoptosis-regulating proteins. Identification of any such cellular apoptosis-regulating proteins which interact with E1B 19 kD protein and with Bcl-2 would enable regulation of apoptosis in cells.